Previous studies from our laboratory demonstrated that a 3 -hydroxysteroid dehydrogenase (3 -HSD) inhibitor, trilostane, in vivo selectively suppressed progesterone, but not estrogen, production by the primate corpus luteum. To determine which substrates are effectively blocked by trilostane, primate luteal cells were incubated in the presence or absence of trilostane or hCG with different steroid substrates. Luteal tissues were collected from monkeys at mid (d 7-8) luteal phase of the menstrual cycle. Collagenase-dispersed (i.e., mixed) cells were sorted by flow cytometry into subpopulations of small and large luteal cells. Cells were incubated in Ham's F10 media for 3h with or without hCG (100ng/ml) or trilostane (250ng/ml) and 1fM steroid substrate (17`-hydroxypregnenolone, 17`-hydroxyprogesterone, dehydroepiandrosterone (DHEA), DHEA-sulfate, or androstandiol). Progesterone (P), androstenedione (A), testosterone (T), estrone (E1), and estradiol (E2) were measured by radioimmunoassay. P production was inhibited by trilostane and enhanced by hCG. None of the steroid substrates affected P production. A and T secretion were greatly increased with DHEA treatment; however, trilostane or hCG had no effect on A or T. E1 production increased with 17`-hydroxypregnenolone and DHEA treatment, but was not affected by trilostane or hCG. E2 secretion was enhanced with DHEA. Additionally, trilostane enhanced E2 production; however, hCG did not. Thus, trilostane is a selective inhibitor of 3 -HSD activity converting pregnenolone to progesterone, but does not block 3 -HSD conversion of the androgens or estrogens further along the steroidogenic pathway, in primate luteal cells.